One-Pot Isothermal Nucleic Acid Amplification Assisted CRISPR/Cas Detection Technology: Challenges, Strategies, and...

The cutting-edge CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas (CRISPR-associated proteins) system, as an emerging molecular diagnostic technique, is driving revolutionary developments in the detection field due to its high specificity and efficiency. However, the CRISPR-based assays typically require the combination with an additional pre-amplification step based on isothermal nucleic acid amplification to meet the requirements of clinical diagnosis, which brings issues including complicated operation and the risk of aerosol contamination. To address these challenges, one-pot CRISPR platforms are emerging as an attractive solution to streamline workflows, enabling rapid, cost-effective, and high-sensitivity diagnostics. This review outlines the current status, challenges, and three key strategies to realize highly efficient one-pot CRISPR-based detection. In addition, further perspectives are outlined that will inspire new exploration and promote one-pot CRISPR/Cas detection as the next generation of diagnostic tools.